1. Field of the Invention
The present invention pertains to the field of protecting groups in organic synthesis and, more particularly, to the use of these compounds as nucleoside protecting groups. Still more specifically the protecting groups are used in the site-specific stepwise synthesis of oligonucleotides.
2. Description of the Prior Art
Deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") are central to information processing in living cells. DNA is the permanent information storage unit, similar to the hard drive on a computer. RNA is the cell's means of transferring and expressing this information from the DNA when necessary, like the RAM in a computer. Both nucleic acid structures are long chain molecules consisting of monomer subunits, the sequence and order of which is the means of coding (FIG. 14), just as the order of letters in a sentence has a specific meaning. There are four monomer building blocks both for DNA and RNA (deoxy-adenosine and riboadenosine are illustrated in FIG. 15). By comparison, the english language uses 26 letters, or building blocks to build a sentence. DNA must be able to store its information permanently and, thus, it is relatively stable. RNA is meant to transfer information temporarily and, thus, is rather easily degraded by enzymes and extreme pH's or other harsh chemical conditions. Although human cells contain .about.6 billion base pairs of deoxyribonucleic acid ("DNA"), it is very useful to synthesize and study oligonucleotides, or short lengths of DNA, of 20-30 bases long. Prior to 1982 the synthesis of oligonucleotides was a rather time consuming process which usually required the skills of chemists. The method of building an oligonucleotide chain requires a series of reactions to elongate the chain one monomer at a time. This series of reactions is repeated for each sequential monomer that must be added to this growing oligonucleotide chain.
Conventional phosphoramidite chemistry, so named for a functional group on the monomer building blocks, was first developed in the early 1980's as disclosed in U.S. Pat. No. 4,415,732. This functional group provided a relatively efficient means of joining a building block monomer to the growing chain. Solid phase synthesis disclosed by Caruthers et al. in U.S. Pat. No. 4,458,066 was another improvement to oligonucleotide synthesis. In this technique, the growing DNA chain is attached to an insoluble support via a long organic linker which allows the the growing DNA chain to be solubilized in the solvent in which the support is placed. The solubilized, yet immobilized, DNA chain is thereby allowed to react with reagents in the surrounding solvent and allows for the easy washing away of the reagents from the solid support to which the oligonucleotide is attached. These significant advances in phosphoramidite chemistry and solid phase synthesis paved the way to making custom DNA synthesis accessible to the average biology lab. Techniques like Sanger sequencing, which relies on synthetic DNA, are essential to the Human Genome Sequencing Project. Other novel techniques, e.g. polymerase chain reaction ("PCR"), have been invented due to the ready availability of synthetic DNA. PCR is one of the principal techniques in forensic testing and DNA fingerprinting.
As can be seen in FIG. 15, there are several sites on the nucleosides of similar chemical nature, e.g. --OH or hydroxyl groups. However, as can be seen In FIG. 14, the monomer subunits in DNA and RNA oligonucleotides must be attached in a site-specific manner. This requires functionalizing a site either on the growing chain or on the incoming base for attachment of the incoming monomer building block to the growing chain. To prevent the incoming monomer from attaching at the wrong site, the wrong sites must be blocked while the correct site is left open to react. This requires the use of what are termed protecting groups. Protecting groups are compounds attached temporarily to a potentially reactive site so as to prevent it from reacting. The protecting group must be stable during said reactions and yet must eventually be removed to yield the original site. The synthesis of oligonucleotides requires several sites to be protected and particular sites must be deprotected while others remain protected. These protecting groups grouped together as a set are termed orthogonal protecting groups.
Phosphoramidite chemistry and solid phase oligonucleotide synthesis protocols use a dimethoxytrityl protecting group for the 5' hydroxyl of nucleosides (see FIG. 15 for numbering 1', 2', 3', 4' and 5'). A phosphoramidite functionality is utilized at the 3' hydroxyl position. Phosphoramidite synthesis generally proceeds from the 3' to the 5' of the ribose or deoxyribose sugar component of the phosphoramidite nucleoside (see FIG. 16 for a schematic representation of this technology). The 5' end of the growing chain is coupled with the 3' phosphoramidite of the incoming base to form a phosphite triester intermediate (the 5' hydroxyl of the added base is protected by a dimethoxytrityl group so only one new base is added to the growing chain at a time). Any unreacted 5' hydroxyls are "capped" off to stop the synthesis of this chain, which would be one base short at the end of synthesis. The triester intermediate is subjected to iodine oxidation after each coupling reaction to yield a more stable phosphotriester intermediate. Without oxidation, the unstable phosphite triester linkage would cleave under the acidic conditions of subsequent synthesis steps. Removal or deprotection of the 5' dimethoxytrityl protecting group of the newly added base is typically accomplished by reaction with acidic solution to yield a free 5' hydroxyl group which can be coupled to the next protected nucleoside phosphoramidite. This process is repeated for each monomer added until the desired sequence is synthesized.
The phosphoramidite technique was a significant advance in the art. Nevertheless, several problems are associated with the phosphoramidite protocols. These problems are as follows. All chemicals used in oligonucleotide synthesis must be compatible with the dimethoxytrityl protecting group, and this circumstance precludes the use of incompatible reagents as well as incompatible protecting groups for protecting the 2' position during RNA synthesis. The removal of the dimethoxytrityl group is reversible and, consequently, the cleaved dimethoxytrityl group must be thoroughly removed from the reaction or quenched to obtain maximum yields. Another problem is the various product impurities due to numerous side reactions that accompany the acid deprotection of the 5' hydroxyl during each cycle of base addition. The acidic conditions of deprotection lead to depurination during synthesis and, particularly, depurination of N-protected adenosine.
Attempts to utilize other protecting groups and reagents has largely failed to overcome the depurination problem. Aprotic acid conditions, which do not lead to depurination, work well only for oligonucleotide sequences less than about twelve units in length. The depurination rate is particularly sensitive to the choice of a protecting group for N-6 of adenosine. Phenoxyacetyl amide protecting groups slow the depurination rate relative to the standard benzoyl group by a factor of about two, but depurination remains a problem for long syntheses and large scale reactions. Amidine protecting groups slow depurination by a significant factor of 20, but the commercial wide spread use of amidines in oligonucleotide synthesis has not transpired, despite being described over 10 years ago.
A significant disadvantage of using a 5' dimethoxytrityl group for the synthesis of oligonucleotides is that such use precludes the synthesis of oligonucleotides having acid labile backbones or functionalities. Modified, or unnatural, backbones are often used to impart stability to oligonucleotides which may be exposed to enzymes which degrade oligonucleotides. For example, antisense oligonucleotides which incorporate diamidates, boranophosphates, or acid labile bases may hold promise in pharmaceutical applications, but present dimethoxytrityl synthesis techniques using the dimethoxytryl protecting group precludes the manufacture of these materials by requiring the use of acid deprotection conditions during oligonucleotide synthesis.
The use of the dimethoxytrityl group further prevents the use of other acid labile protecting groups. This is important for RNA synthesis because another hydroxyl group at the 2' position (see FIG. 15) must be protected. The use of the dimethoxytrityl group at the 5' position therefore prevents the successful use of acid labile groups for 2' protection during RNA synthesis.
RNA is more difficult to synthesize relative to DNA. This is not only due to the need for an additional orthogonal protecting group that must be compatible with all other protecting groups but also because of the instability of RNA as mentioned earlier. The significance of this, relative to RNA oligonucleotide synthesis, is that the 2' hyxdroxyl protecting groups preferably should be the last protecting groups removed and must be done so under mild, non-degradative conditions.
RNA is particularly difficult to synthesize with the conventional 5' dimethoxytrityl protecting group due to the difficulty of finding a suitable 2' protecting group. A t-butyldimethylsilyl ether protecting group is presently used at the 2' position in conjunction with the 5' dimethoxytrityl group, but the process lacks reliability. Coupling yields are poor and high pressure liquid chromatography ("HPLC") analysis of RNA product shows significant unexplained impurities. These impurities significantly increase the difficulty of isolating the desired oligonucleotide. Complete deprotection of the t-butyidimethylsilyl group from the oligonucleotide is questionable for long sequences. Deprotection is done in organic solvents which further complicates the isolation of the water soluble RNA from the deprotection reaction. Synthesis of the protected ribonucleosides used for oligonucleotide synthesis is also rather costly and prone to yield impurities that complicate synthesis of natural RNA. As a result of all these complications, the use of a 5' dimethoxytrityl group in conjunction with a 2' t-butyidimethylsilyl protecting group is not reliable or efficient.
Another choice for 2' protection is the acetal class of protecting groups. They can be removed under mild aqueous acidic conditions. This is desirable as a final RNA deprotection step because RNA is soluble in water, is easily isolated from water and is only slightly unstable under conditions for deprotection of some of the more labile acetals. The use of acetals at the 2' hydroxyl position has been attempted in conjunction with a 5' dimethoxytrityl group, but very stable acetals must be used with the acid labile dimethoxytrityl group. Removal of acetals that are stable under dimethoxytrityl deprotection conditions requires strong acidic conditions that degrade the RNA. Acetals have been attempted with alternative 5' protecting groups which are deprotected under non-acidic conditions, e.g. leuvinyl, but without significant success.
Oligonucleotide synthesis according to H-phosphonate protocols will permit a single oxidation step at the conclusion of the synthesis cycles. However, coupling yields are less efficient than those for phosphoramidite chemistry and oxidation requires longer times and harsher reagents than amidite chemistry.
There remains a need to find an alternative 5' protecting group that permits the use of acid labile linkages, reagents and protecting groups that are incompatible with dimethoxytrityl, increased process yields, and reduction of product impurities without significant increase in cycle times. An alternative 5' protecting group would further be a significant advantage for RNA synthesis by allowing the use of more suitable 2' protecting groups.